Interpretive Summary: Two genes that cause a defect in Salmonella colonization of alfalfa sprouts were characterized. The data suggest that swarming, movement across surfaces, is important for sprout colonization by Salmonella.

Technical Abstract:
Incidences of bacterial foodborne illness caused by ingestion of fresh produce are rising. Instead of being a case of incidental contamination, the animal pathogen Salmonella enterica utilizes specific molecular mechanisms to attach to and colonize plants. This work characterizes two S. enterica genes of unknown function; a putative periplasmic protein, STM0278, and putative protein with a hydrolase in the C-terminus, STM0650. STM0278 and STM0650 are important for seedling colonization but have different roles throughout the continuum of colonization. Both mutants were reduced for colonization of alfalfa seedlings at 24 h and the STM0278 mutant was also reduced at 48 h. Both genes are expressed in planta at 4 h following inoculation of 3-day-old seedlings and 72 h after seed inoculation. This suggests that the role of STM0650 in seedling colonization is less important later or duplicated by other mechanisms. Mutants of STM0278 and STM0650 are defective in swarming. The STM0278 mutant fails to swarm in 24 h while the STM0650 mutant is delayed. Addition of surfactant restores a STM0278 mutant suggesting that STM0278 is involved in surfactant or osmotic agent production or deployment. Alfalfa seed exudates as the sole nutrient source were capable of perpetuating S. enterica swarming. Sequence analysis revealed homologous sequences of STM0278 and STM0650 in plant-associated bacteria, but none in E. coli. Phylogenetic analysis of STM0650 showed similar sequences from diverse classes of plant-associated bacteria. Bacteria that preferentially colonize roots, including S. enterica, may use a similar hydrolase for swarming or biofilm production on plants. Multicellular behaviors by S. enterica appear central to plant colonization. S. enterica genes involved in plant colonization and survival outside of a host are most likely among the function unknown.